Although increased osteoclastic bone resorption is the mechanism responsible for bone loss in the postmenopausal period, in malignant diseases which involve the skeleton and Paget's disease, the molecular mechanisms responsible for osteoclastic bone resorption remain unclear. Recently, new light has been shed on this process with the observation that expression of the src proto-oncogene (pp6Ocsrc) is required (and essential) for normal osteoclastic bone resorption. This was shown unexpectedly in transgenic mice deficient in src expression and produced by the technique of introducing the null mutation into the gene by homologous recombination. Our plan In this application is to examine the mechanisms responsible for this impaired osteoclast function using in vitro and in vivo techniques in src deficient (src-) and normal wild type mice. We plan to determine whether src expression is required for osteoclast formation, or activation of cells already formed, or both. To evaluate this question, we plan to culture bone marrow cells and spleen cells from homozygous src deficient mutants. We also plan to determine whether the osteopetrosis is due to a microenvironmental defect or to a defect in cells in the osteoclast lineage. Two approaches will be utilized in these experiments. In one approach, experiments are currently being performed by Dr. Soriano using transplants of fetal liver cells to lethally irradiated recipients. In a second approach, we will perform experiments in vitro using spleen cells or bone marrow cells (as a source of osteoclast precursors) co-cultured with stromal cells which are obtained either from cultures from the marrow cavity or from freshly isolated calvarial cells. These experiments should allow us to determine whether src expression is required in cells in the osteoclast lineage or the accessory cells involved in bone resorption. We also plan to examine the effects of inhibitors of src tyrosine kinase on osteoclastic bone resorption both in vitro and in vivo. We have a number of inhibitors available to us, one of which preferentially inhibits src tyrosine kinase. In addition, we plan to examine the effects of parathyroid hormone and other stimulators of bone resorption on the expression of tyrosine kinase activity, the phosphorylated substrate of tyrosine kinase, and src expression in isolated osteoclasts, osteoblasts, and accessory cells associated with bone resorption. These experiments should enable us to determine a) whether the requirement for src expression is in the osteoclast lineage or in accessory cells; b) whether src expression is required for formation of osteoclasts, or for their activation; c) whether PTH exerts its effects on bone resorption to enhance expression of src tyrosine kinase; and d) exploration of the potential of novel approaches of inhibiting bone resorption both in vitro and in vivo.